PCDH15 dual-AAV gene therapy for deafness and blindness in Usher syndrome type 1F models
Maryna V. Ivanchenko, … , Bence György, David P. Corey
Maryna V. Ivanchenko, … , Bence György, David P. Corey
Published October 23, 2024
Citation Information: J Clin Invest. 2024;134(23):e177700. https://doi.org/10.1172/JCI177700.
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PCDH15 dual-AAV gene therapy for deafness and blindness in Usher syndrome type 1F models

Abstract

Usher syndrome type 1F (USH1F), resulting from mutations in the protocadherin-15 (PCDH15) gene, is characterized by congenital lack of hearing and balance, and progressive blindness in the form of retinitis pigmentosa. In this study, we explore an approach for USH1F gene therapy, exceeding the single AAV packaging limit by employing a dual–adeno-associated virus (dual-AAV) strategy to deliver the full-length PCDH15 coding sequence. We demonstrate the efficacy of this strategy in mouse USH1F models, effectively restoring hearing and balance in these mice. Importantly, our approach also proves successful in expressing PCDH15 protein in clinically relevant retinal models, including human retinal organoids and nonhuman primate retina, showing efficient targeting of photoreceptors and proper protein expression in the calyceal processes. This research represents a major step toward advancing gene therapy for USH1F and the multiple challenges of hearing, balance, and vision impairment.

Authors

Maryna V. Ivanchenko, Daniel M. Hathaway, Eric M. Mulhall, Kevin T.A. Booth, Mantian Wang, Cole W. Peters, Alex J. Klein, Xinlan Chen, Yaqiao Li, Bence György, David P. Corey

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Figure 1

Splicing and protein production in vitro.

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Splicing and protein production in vitro. (A) Recombination and splicing...
(A) Recombination and splicing of dual vectors to produce full-length protein. The reassembly is mediated by non-homologous end joining of the inverted terminal repeats (ITRs) and/or homologous recombination of the highly recombinogenic (AK) sequence. After transcription, splicing can occur from the splice donor site (SD) in vector 1 to the splice acceptor site (SA) in vector 2, creating a full-length Pcdh15 mRNA. (B) mRNA was obtained from HEK cells and reverse-transcribed (n = 5). A cDNA library was Sanger-sequenced around the splice junction, confirming proper recombination and splicing. (C) Dual AAVs (encoding PCDH15 with or without an N-terminal HA tag) were added to HEK293 cell cultures (n = 3 per group). Immunostaining was performed with anti-PCDH15 or anti-HA antibodies (magenta). Representative confocal images demonstrate normal trafficking of PCDH15, either with or without the N-terminal HA tag, to the cell membrane after dual-AAV delivery. Labeling was similar to that with transfection of a single plasmid encoding full-length PCDH15. No specific signal was detected in the control samples (n = 3 per group). Scale bar: 10 μm. CDS, coding sequence; CMV, cytomegalovirus; DIC, differential interference contrast; IRES, internal ribosome entry site; polyA, polyadenylation signal; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element.